Methods for quantifying assay material contained in a biological sample include methods in which material in an assay sample is agglutinated using a substance to induce an antigen-antibody reaction with the assay material, and the concentration of the assay material is calculated based on the degree of agglutination. Examples of such methods include immunoturbidity, immunonephelometry, counting immunoassay (CIA) and the like.
A calibration curve, which represents the relationship between the concentration of the assay material and information reflecting the degree of agglutination of the material in the assay sample, is used when calculating the concentration of the assay material by these methods. FIG. 14 shows an example of a calibration curve when the information reflecting the degree of agglutination of carrier particles is the absorbance in the immunoturbidity method using carrier particles. In FIG. 14, when the concentration of the assay material is in the low concentration range, the absorbance gradually increases in conjunction with the increase in the concentration of the assay material. However, above a certain concentration of the assay material (high concentration range), there is a reduction in the absorbency (referred to as a ‘zone phenomenon’). As shown in FIG. 14, when the zone phenomenon occurs, two concentrations (C1 and C2) are obtained in the low concentration range and high concentration range of a single absorbency level A, such that the concentration of the assay material cannot be ultimately determined. Therefore, the concentration range in which the assay material can be measured is limited.
Japanese Laid-Open Patent Publication No. 61-280568 discloses a method for measuring an assay material even when the specimen contains a high concentration of assay material. This method utilizes CIA using carrier particles as the principle of measurement. CIA first mixes a specimen including an assay material and carrier particles on which antibody or antigen against the assay material is immobilized, and agglutinates the carrier particles by an antigen-antibody reaction. After a predetermined reaction time, the degree of agglutination is detected and the carrier particle distribution is obtained, then the degree of agglutination of the carrier particles is analyzed from the particle distribution, and the concentration of the assay material is calculated based on the degree of agglutination. The predetermined reaction time in this case is the time interval from the initiation of the antigen-antibody reaction until agglutination is detected.
In the method disclosed in Japanese Laid-Open Patent Publication 61-280568, a calibration curve is prepared across the entire region including the low concentration range and high concentration range, and the agglutination of the carrier particles is determined for the reaction time T1 and reaction time T2 of the antigen-antibody reaction. Then, a concentration is calculated from the degree of agglutination and calibration curve at reaction time T1, and another concentration is calculated from the degree of agglutination and calibration curve at reaction time T2. In this way the concentrations are compared for the reaction time T1 and reaction time T2, and a concentration common to both reaction time T1 and reaction time T2 is designated as the final assay material concentration.
The method of Japanese Laid-Open Patent Publication No. 61-280568 is described below using FIG. 15. FIG. 15 shows the calibration curves at reaction time T1 and reaction time T2 (where T1<T2). In FIG. 15, calibration curve T1 is the calibration curve at reaction time T1, and calibration curve T2 is the calibration curve at reaction time T2. For example, when the agglutination at reaction time T2 is designated B, the concentration of the assay material is either C1 or C2. Likewise, when the agglutination at reaction time T1 is A1, the concentration C1, which is common to each reaction time on the calibration curves, becomes the concentration of the assay material in the specimen. Similarly, when the agglutination at reaction time T1 is A2, the concentration C2, which is common to each reaction time on the calibration curves, becomes the concentration of the assay material in the specimen.
However, the value of the degree of agglutination at reaction time T2 is indispensable for determining the concentration of the assay material in this method. That is, the degree of agglutination at both reaction times T1 and T2 are invariably required in this method.